The goal of this research is the elucidation of the reaction mechanism of the Na, K-ATPase, in order to define the molecular processes involved in ion transport by this enzyme. Physical-biochemical studies on a purified enzyme preparation will concentrate on two approaches. (1) Spectroscopic studies, using fluorescent probes, intrinsic protein flourescence, and difference ultraviolet spectroscopy, are directed at distinguishing specific conformational states of the enzyme resulting from particular ligand-enzyme interactions. The rates of formation and breakdown of these conformational states will be determined using stopped-flow and rapid-mixing techniques, and these rates will be correlated with the kinetic parameters of the enzymatic reaction. In addition, nano-second flourescence lifetime measurements are proposed in order to study changes in distances between fluorophores on the enzyme (by energy-transfer measurements) and alterations in enzyme volume and shape (by time-dependent fluorescence depolarization techniques) induced by ligand binding. (2) Chemical modifications of essential amino acids with group-specific reagents and site-directed inhibitors (affinity labels) are designed to examine the amino acids participating in the reaction process and to determine their role. The relationship between the number of groups reacting and the loss of activity will be investigated, and the modified residues identified spectroscopically and by amino acid analysis. The modified enzyme will be examined in terms of changes in kinetic parameters and in spectroscopic properties.